Chemotherapy Drugs: Less is More

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1 Chemotherapy Drugs: Less is More Background BY: Noorain Shethwala and Sophia Oke Since chemotherapy was first recognized as a proven treatment for cancer, it has been the subject to intense scientific scrutiny. Chemotherapy refers to the treatment of tumours through the introduction of chemicals designed to kill rapidly growing (cancerous) cells. Often, one major downside to chemotherapy treatments is the everpresent side effects, and the possible return of the cancer. One of the most devastating forms of cancer, and the one that most affects people worldwide is breast cancer. Breast cancer is usually caused by defective inherited genes such as BAX, BRCA 2 and p53. The BAX gene, induces apoptosis by traveling to the surface of the mitochondria of cells where it triggers the release of cytochrome c; ultimately binding to Apaf-1protein (Apoptotic Protease Activating Factor-1). Genes such as BAX are referred to as tumour suppressors as they help control tumour growth. We explored the idea of using BAX as a chemosensitizer, which is a drug that makes tumours more sensitive to the effects of chemotherapy. We also explored the use of a virotherapy, specifically, adenovirus - 4EGFP as a chemosensitizer. Purpose The objective of our experiment was to reduce the quantity of drug generally given to patients by making the cells more chemosensitive and prone to apoptosis through their treatment with -4EGFP adenovirus or pegfp-bax plasmid. With a reduction in the amount of chemotherapy drug used, there would be a reduction of side effects to chemotherapy experienced by patients (due to lower dosage). We used two colon cancer cell lines: one with only one mutant allele, BAX (+/-), and another with two mutant, BAX (-/-), to asses the impact to both. BAX (+/-) has one portion of the functional BAX gene, whereas BAX (-/-) lacks the entire functional BAX genes.

2 Hypothesis The quantity of Chemotherapy Drug administered to patients can be reduced by making cancerous cells more prone to apoptosis with the use of -4EGFP adenovirus and pegfp-bax plasmid. Procedure Our experiment was conducted with the usage of two different genotypes of the colon cancer cell cultures HCT 116, BAX (+/-) and BAX (-/-). Both cell lines were infected with the chemotherapy drugs at different concentrations (1uM, 2uM and 375uM). Along with the drug, an MOI (Multiplicity of Infection): 5 was used for the adenovirus. We used the BAX gene carried by a plasmid, as it functions in a key role in inhibiting the growth of tumour cells, and is lacking in our 2 cell lines. We had an untreated control present along with all of our cell samples of drug and drug with plasmid in an effort to compare cell viability between the different samples. We performed the same procedure for our adenovirus. In order to keep our cancerous cells alive for the duration of our experiment, we passed the cells every 2 days following the cell passage protocol below. Cell Passage Protocol: 1. From a donor flask, remove the old media and wash cells with PBS. 2. Add 1X Trypsin, ph 7.4 and incubate at 37ºC for 2 mins. 3. Add DMEM (media with 1% FBS) directly to the cells and pipette gently. 4. Pass the cells to new flasks or plates (35 mm plates). Results Preliminary results were mainly qualitative as we assessed cell structure before performing cell counts. Initially, after testing all three chemotherapy drugs (Indomethacin, Cyclohexamide, Sulindac), we concluded that Indomethacin would be the most effective chemotherapy drug to be used in our experiments as it was the fastest operating. In our untreated control we observed healthy, medium confluency cells, rapidly growing and as we increased the concentration of Indomethacin we began to see far fewer, apoptotic cells bearing a cloud like formation.

3 pegfp- BAX Plasmid Transfection Results: (Counted at, 24, 48 Hour Periods), 48 Hours Post Transfection: BAX (+/-) Genotype, 48 Hours Post Transfection: BAX (-/-) Genotype Figure 3a): Cell Death after 48 hours of BAX Plasmid Transfection + 25 Indomethacin Figure 3b): Cell Death 48 hours after Plasmid Transfection in BAX (-/-) Genotype Figure 1a) Cell Death BAX (+/-) Figure 1b) Cell Death BAX (-/-) D e a d C e ll C o n c e n tr a tio n (p e r 1 m m x 1 m m s qua r e ) Time Period Control (+/-) Plasmid + 25ul Indo 25 Indo Dead Cell count (per 1mm x 1mm square) Time Period Contol (-/-) Plasmid + Indo 25ul 25 ul Indo As shown in the graph, there was minimal difference in cell death due to the addition of the BAX plasmid in the BAX (+/-) genotype As shown in the graph above, there was substantial difference in cell death due to the addition of the BAX plasmid in the BAX (-/-) genotype. Thus Plasmid transfection was effective. As seen in the graphs above, as well as microscopic pictures of cell structure, we were able to conclude that the addition of a functional BAX gene along with a medium (25 um) concentration of Indomethacin proved to be more effective in killing cancerous cells than just the drug alone. Overall, we noticed a 22% increase in cell death in the BAX (-/-) due to the addition of the plasmid, thus proving this form of treatment more effective in the BAX (-/-) genotype. -4EGFP Adenovirus Infection Results: (Counted at, 48 Hours) D e a d C e ll C o u n t ( p e r 1 m m x 1 m m s q u a r e ) Figure 4b): Adenovirus Infection Figure 2: Adenovirus Infection (+/-) MOI: (+/-) 375 (+/-) Experiments (-/-) MOI: (-/-) (-/-) 25 hours 48 hours Upon deciding Indomethacin would be used we performed repeated tests with the combination of Adenovirus (at MOI: 5) and Indomethacin at concentrations 1uM, 25uM, and 375uM. After 3 trials, the adenovirus proved to consistently assist in the apoptosis among these concentrations. The adenovirus was particularly effective in the BAX (+/-) genotype and had a significant 8% increase in the amount of dead cells from just 25 um Indomethacin to 25uM and adenovirus.

4 Conclusions After many experiments, pictures, and counts, we concluded that our hypothesis was correct. The combination of adenovirus and Indomethacin drug treatment showed increased cell death compared to drug alone. Similarly, a combination of BAX expressing plasmid transfection showed higher levels of cell death compared to drug alone. Our results demonstrated that a lower chemotherapy drug amount can be used as long as it is co-administered with a plasmid or adenovirus. From our preliminary experiments, the BAX plasmid was most effective in cell killing of the BAX (-/-) genotype cells, and the adenovirus was most effective in the BAX (+/-). All of our experiments were performed with three trials, however, to attain more accurate results this experiment should be repeated more times. Based on these preliminary experiments using chicken adenovirus and a BAX plasmid to treat cancer cells, we feel that through our project we have discovered new ground for scientific research to further develop this concept and perhaps provide a novel approach in which lower levels of chemotherapeutic drugs would be needed to control a cancer. Acknowledgements We are profoundly grateful to Dave Leishman, our lab technician, who worked with us everyday for months until we finally finished performing tests. We greatly appreciate his time and effort in providing us with resources and teaching us vital skills. We would also like to thank our mentor, Dr. Peter Krell, for his untiring support and motivation. He constantly offered us improvement ideas and told us how to perceive our results. We would like to thank them for their dedication and faith in us. Dr. Mosser generously provided us with our cells and plasmid for which we are immensely grateful. As well, a special thanks goes out to Ms. Brown and Mr. Gajic who found mentors for us, and provided us with any additional information we needed. Bibliography Nechushtan, Amotz. "Conformation of the Bax C-terminus regulates subcellular location and cell death." The EMBO Journal 18 (1999):

5 Zhang, Lin. "Anticancer Agents Role of BAX in the Apoptotic Response." USB Science AAS (2): 29, Feb. 29 < National Institute of Health. (27, Sep ). Breast Cancer., Retrieved Nov 9, 28, from Mei, J. Overexpression of PTEN in Breast Cancer Cells and Normal Cells. Retrieved Nov 4, 25, from Wang et al. (22, Mar 1). Plasmid 16246: pcdna3 236HSC WT (BRCA2). Retrieved Nov 15, 28, from Unknown. Chemotherapeutic agents/antineoplastic agents. Retrieved Nov 15, 25, from Lowe, Scott. "p53-dependent Apoptosis Modulates the Cytotoxicity of Anticancer Agents." Cell. Cell Press (1993):